Idiopathic pulmonary fibrosis (IPF) is a disease of aging. IPF carries a high morbidity and mortality, with a median survival rate of less than three years. The incidence and prevalence of IPF increase drastically with age; however, despite this strong association, cellular/molecular mechanisms that account for the aging predilection to fibrotic disease have not been elucidated. Recent studies from our laboratory have identified a reactive oxygen species (ROS)-generating enzyme, NADPH oxidase-4 (Nox4), in mediating differentiation of fibroblasts (Fbs) to myofibroblasts (MFbs), key effectors of fibrogenesis, and in in-vivo lung fibrosis in murine models of lung injury. Our preliminary studies indicate that the biological actions of Nox4 may be modulated by the expression of SIRT3, a mitochondrial sirtuin. Both cellular senescence and TGF-?1 mediate suppression of SIRT3; our preliminary studies support a role for epigenetic silencing of SIRT3 involving both DNA methylation and histone modification. Decreased expression of SIRT3 promotes a senescent and pro-fibrotic Fb phenotype. TGF-?1-induced down-regulation of SIRT3 is associated with hyper-acetylation of mitochondrial proteins, supporting a role for altered mitochondrial bioenergetics in MFbs. Human subjects with IPF express low levels of SIRT3 in myofibroblastic foci (by immunohistochemistry), as well as in ex-vivo Fbs isolated from IPF lungs. We have developed a novel aging model of non-resolving fibrosis in mice; fibrosis in young mice (2 months) resolves by >50% by 4 months post-bleomycin, whereas aged mice (18 months) show persistent fibrotic response. While SIRT3 levels decrease during the fibrogenic phase in both groups, young mice demonstrate a capacity to recover SIRT3 levels during resolution; in contrast, aged mice manifest sustained down-regulation of SIRT3. The central hypothesis to be tested in this grant proposal is that, in the context of aging, lung injury results in sustained, epigenetically-regulated SIRT3 silencing that leads to mitochondrial dysfunction, MFb senescence and apoptosis resistance, leading to persistent fibrosis with aging. Our specific aims are to: (1) determine epigenetic mechanisms for SIRT3 down-regulation with cellular senescence and with TGF-?1 signaling in lung Fbs; (2) determine the role of SIRT3 in regulating mitochondrial bioenergetics, Fb senescence and apoptosis resistance; (3) determine whether whole-animal SIRT3 knockout and/or conditional genetic deletion of SIRT3 in collagen-producing/mesenchymal cells induce(s) persistent fibrosis in young mice. The completion of the Aims in this proposal will: (a) elucidate epigenetic mechanisms that control SIRT3 expression with cellular senescence/aging; (b) provide mechanistic insights into the role of SIRT3 in maintenance of mitochondrial bioenergetics and cellular plasticity/fate; (c) provide proof-of-concept that SIRT3 induction in the context of age-associated fibrosis facilitates fibrosis resolution, uncovering a novel therapeutic approach to non-resolving fibrotic disorders such as IPF.